Identification of two regions in the p140Cap adaptor protein that retain the ability to suppress tumor cell properties

p140Cap is an adaptor protein that negatively controls tumor cell properties, by inhibiting in vivo tumor growth and metastasis formation. Our previous data demonstrated that p140Cap interferes with tumor growth and impairs invasive properties of cancer cells inactivating signaling pathways, such as the tyrosine kinase Src or E-cadherin/EGFR cross-talk. In breast cancer p140Cap expression inversely correlates with tumor malignancy. p140Cap is composed of several conserved domains that mediate association with specific partners. Here we focus our attention on two domains of p140Cap, the TER (Tyrosine Enriched Region) which includes several tyrosine residues, and the CT (Carboxy Terminal) which contains a proline rich sequence, involved in binding to SH2 and SH3 domains, respectively. By generating stable cell lines expressing these two proteins, we demonstrate that both TER and CT domains maintain the ability to associate the C-terminal Src kinase (Csk) and Src, to inhibit Src activation and Focal adhesion kinase (Fak) phosphorylation, and to impair in vitro and in vivo tumor cell features. In particular expression of TER and CT proteins in cancer cells inhibits in vitro and in vivo growth and directional migration at a similar extent of the full length p140Cap protein. Moreover, by selective point mutations and deletion we show that the ability of the modules to act as negative regulators of cell migration and proliferation mainly resides on the two tyrosines (Y) inserted in the EPLYA and EGLYA sequences in the TER module and in the second proline-rich stretch contained in the CT protein. Gene signature of cells expressing p140Cap, TER or CT lead to the identification of a common pattern of 105 down-regulated and 128 up-regulated genes, suggesting that the three proteins can act through shared pathways. Overall, this work highlights that the TER and CT regions of p140Cap can efficiently suppress tumor cell properties, opening the perspective that short, defined p140Cap regions can have therapeutic effects

The scaffold protein p140Cap limits ERBB2-mediated breast cancer progression interfering with Rac GTPase-controlled circuitries.

The docking protein p140Cap negatively regulates tumour cell features. Its relevance on breast cancer patient survival, as well as its ability to counteract relevant cancer signalling pathways, are not fully understood. Here we report that in patients with ERBB2-amplified breast cancer, a p140Cap-positive status associates with a significantly lower probability of developing a distant event, and a clear difference in survival. p140Cap dampens ERBB2- positive tumour cell progression, impairing tumour onset and growth in the NeuT mouse model, and counteracting epithelial mesenchymal transition, resulting in decreased metastasis formation. One major mechanism is the ability of p140Cap to interfere with ERBB2- dependent activation of Rac GTPase-controlled circuitries. Our findings point to a specific role of p140Cap in curbing the aggressiveness of ERBB2-amplified breast cancers and suggest that, due to its ability to impinge on specific molecular pathways, p140Cap may represent a predictive biomarker of response to targeted anti-ERBB2 therapies

Mapping of p140Cap phosphorylation sites: the EPLYA and EGLYA motifs have a key role in tyrosine phosphorylation and Csk binding, and are substrates of the Abl kinase.

Protein phosphorylation tightly regulates specific binding of effector proteins that control many diverse biological functions of cells (e. g. signaling, migration and proliferation). p140Cap is an adaptor protein, specifically expressed in brain, testis and epithelial cells, that undergoes phosphorylation and tunes its interactions with other regulatory molecules via post-translation modification. In this work, using mass spectrometry, we found that p140Cap is in vivo phosphorylated on tyrosine (Y) within the peptide GEGLpYADPYGLLHEGR (from now on referred to as EGLYA) as well as on three serine residues. Consistently, EGLYA has the highest score of in silico prediction of p140Cap phosphorylation. To further investigate the p140Cap function, we performed site specific mutagenesis on tyrosines inserted in EGLYA and EPLYA, a second sequence with the same highest score of phosphorylation. The mutant protein, in which both EPLYA/EGLYA tyrosines were converted to phenylalanine, was no longer tyrosine phosphorylated, despite the presence of other tyrosine residues in p140Cap sequence. Moreover, this mutant lost its ability to bind the C-terminal Src kinase (Csk), previously shown to interact with p140Cap by Far Western analysis. In addition, we found that in vitro and in HEK-293 cells, the Abelson kinase is the major kinase involved in p140Cap tyrosine phosphorylation on the EPLYA and EGLYA sequences. Overall, these data represent an original attempt to in vivo characterise phosphorylated residues of p140Cap. Elucidating the function of p140Cap will provide novel insights into its biological activity not only in normal cells, but also in tumors

Integrins and signal transduction

Integrin signaling has a critical function in organizing cells in tissues during both embryonic development and tissue repair. Following their binding to the extracellular ligands, the intracellular signaling pathways triggered by integrins are directed to two major functions: organization of the actin cytoskeleton and regulation of cell behaviour including survival, differentiation and growth. Basic research conducted in the past twelve years has lead to remarkable breakthroughs in this field. Integrins are catalytically inactive and translate positional cues into biochemical signals by direct and/or functional association with intracellular adaptors, cytosolic tyrosine kinases or growth factor and cytokine receptors. The purpose of this chapter is to highlight recent experimental and conceptual advances in integrin signaling with particular emphasis on the ability of integrins to regulate Fak/Src family kinases (SFKs) activation and the cross-talk with soluble growth factors receptors and cytokines

Phosphopeptides of p140Cap protein.

<p>Phosphopeptides of p140Cap protein.</p

Prediction of tyrosine phosphorylation and relative score.

<p>Prediction of tyrosine phosphorylation and relative score.</p

p140Cap tyrosine phosphorylation on EPLYA and EGLYA sequences regulate Csk binding.

<p><b>A.</b> cDNAs encoding GFP, GFP-p140Cap full length (p140 WT) and its double mutant (p140 EPL<b>Y/F</b>A, EGLY<b>/F</b>A) were used to transfect HEK-293 cells. Cells were treated with 100 micromolar pervanadate solution as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054931#pone-0054931-g002" target="_blank">Figure 2B</a> and extracts were immunoprecipitated with a specific antibody to p140Cap and analysed by western blotting using monoclonal antibodies PY99, p140Cap, and Csk. The results are representative of six independent experiments. <b>B.</b> HEK-293 cells transfected as in A for 48 hours, were starved overnight and treated for 0, 5, 15 minutes with 20%FBS. Cell extracts were immunoprecipitated with a specific antibody to p140Cap. Immunocomplexes were analysed by western blotting using monoclonal antibodies specific for phosphotyrosines, p140Cap, Csk and Vinculin.</p

Identification of Abl as the tyrosine kinase responsible of p140Cap tyrosine phosphorylation on EPLYA and EGLYA sequences. A–B.

<p>For each recombinant kinase, bar charts of the mean values of the triplicate activity raw counts of kinase activity and the means of the corresponding background values of the synthetic peptides with (black bars) or without (white bars) enzyme are indicated. The synthetic peptides contain respectively EPLYA (A) and EGLYA (B) sequences. A. cDNAs encoding GFP and GFP-p140Cap full length (p140 WT) were used to transfect HEK-293 cells. After 24 hours, cells were starved and treated with 10 micromolar Src inhibitor SU6656 or Abl inhibitor Imatinib for 16 hours. Cell extracts were immunoprecipitated with a specific antibody to p140Cap and analysed by western blotting using monoclonal antibodies for phosphotyrosine and p140Cap. <b>B.</b> Left panel. cDNAs encoding GFP, GFP-p140Cap full length (p140 WT) and its double mutant (p140 EPLY/FA, EGLY/FA) were used to transfect HEK-293 cells together with cDNA encoding for active BCR-Abl. Extracts were immunoprecipitated with a specific antibody to p140Cap and analysed by western blotting using monoclonal antibodies to phosphotyrosines (PY99) and p140Cap. Right panel. cDNAs encoding GFP, GFP-p140Cap full length (p140 WT) and its double mutant (p140 EPLY/FA, EGLY/FA) were used to transfect HEK-293 cells. Cells were treated with 100 micromolar pervanadate solution for five minutes and extracts were processed as in the left panel.</p

p140Cap tyrosine phosphorylation depends mainly on two tyrosine residues. A.

<p>Schematic representation of p140Cap structure and localization of FYELE, EPLYA, EGLYA, and ADPYG sequences into the tyrosine rich region. These four tyrosine residues have been mutated to phenylalanine. <b>B.</b> cDNAs encoding GFP, GFP-p140Cap full length (p140 WT) and its single (p140 EPL<b>Y/F</b>A, p140 EGL<b>Y/F</b>A), double (p140 EPL<b>Y/F</b>A, EGL<b>Y/F</b>A; p140 F<b>Y/F</b>ELE, ADP<b>Y</b>/<b>F</b>G) and triple (p140 EPL<b>Y/F</b>A, EGL<b>Y/F</b>A, ADP<b>Y</b>/<b>F</b>G) mutants were used to transfect HEK-293 cells. 48 hours after transfection cells were treated for 5 minutes with 100 micromolar pervanadate solution. Cell extracts were immunoprecipitated with a specific antibody to p140Cap and immunocomplexes were analysed by western blotting using monoclonal antibodies to phosphotyrosine (PY99), p140Cap and Tubulin respectively. The results are representative of six independent experiments.</p